胸膜肺炎放线杆菌Lip40蛋白生物学特性研究
发布时间：2018-08-24 16:34
【摘要】：胸膜肺炎放线杆菌(A ctinobacillus pleuropneumoniae, APP)是引起猪胸膜肺炎(Porcine pleuropneumonia)的病原菌,该病是一种严重的猪呼吸道疾病且具有致死性,给养猪业造成了极大的经济损失。到目前为止,已经发现了15种APP血清型。APP的感染以及致病过程中有许多毒力因子的参与,包括RTX毒素、荚膜多糖、脂多糖、粘附因子、蛋白酶、外膜蛋白以及转录调控因子等。本实验通过多种生物信息学分析软件,预测发现APP中共有60种脂蛋白,并从中挑选出一个具有特殊串联重复序列Q(E/D/P)QPK的脂蛋白Lip40。统计发现不同血清型APP菌株中,该重复序列的重复次数不同。通过BioEdit (7.0 版)对Lip40蛋白进行多序列比对分析发现,其与多种已知的脂蛋白和Tbp蛋白具有较高的相似性|。利用SWISS-model对其三维结构进行预测分析,结果显示Lip40蛋白由1个p筒(该β筒由8条p折叠构成)以及4条p折叠所构成,并通过WinCoot软件将Lip40蛋白分别与APP TbpB N lobe、C lobe进行三维结构重叠比对,发现Lip40蛋白与两者的三维结构都具有较高的相似性。为了获得重组Lip40蛋白,本研究首先通过PCR克隆到APP SLW01菌株中的lip40基因,目的基因经限制性内切酶酶切后,连入pGEX-KG载体,转化E.coli BL21感受态细胞,经IPTG诱导获得重组蛋白rLip40。经免疫印迹分析,rLip40泳道上出现一个分子量约为56 kDa的杂交带,表明抗APP的阳性血清可以特异性的识别并结合rLip40蛋白,说明rLip40蛋白具有较好的免疫反应性。另外,本研究以rLip40蛋白为免疫原免疫大白兔,获得兔抗rLip40多克隆抗体,其效价可达1：2×105,表明rLip40蛋白可以诱导动物机体产生较强的免疫反应,说明Lip40蛋白具有良好的免疫原性。为鉴定Lip40蛋白的亚细胞定位,本研究分别提取APP各亚细胞蛋白组分,以兔抗rLip40蛋白多克隆抗体为一级抗体,通过免疫印迹方法对Lip40蛋白进行亚细胞定位,结果发现其定位于外膜,与之前生物信息学结果相同。之后本研究分析了Lip40蛋白在不同应激条件下的表达情况,结果显示在高温、厌氧和低温等应激条件下,Lip40蛋白在转录水平上和翻译水平上均呈现上调,故认为Lip40蛋白是一个多重应激响应因子,并推测Lip40蛋白可能是APP的毒力因子,可能参与病原菌-环境以及病原菌-宿主之间的相互作用。本研究分析了Lip40蛋白的免疫保护性,以rLip40蛋白为免疫原,免疫小鼠,随后以致死剂量的高致病性APP菌株攻毒,结果表明Lip40蛋白对小鼠的保护率达到75%,虽然其保护率低于ApxIA毒素蛋白(100%)和灭活疫苗(91.7%),但显著高于空白对照组。因此,Lip40蛋白可以作为高效APP疫苗研发的候选蛋白。本研究还以前期构建的lip40基因缺失突变株△lip40为亲本,成功构建了回复突变株CΔlip40,并对其生物学特性进行了初步分析。穿梭质粒pJFF-lip40可在APP中稳定遗传,并且未对APP生长能力造成显著影响。此外,APP SLW01菌株和△lip40对氯霉素高度敏感,但回复突变株CAlip40较前两种菌株对氯霉素抗性明显增强,表明穿梭质粒pJFF224-XN抗性基因可在APP菌株中稳定表达。回复突变株CΔlip40的成功构建为分析Lip40蛋白的致病机制提供了有效材料,本实验中建立的APP互补菌株构建系统以及APP抗性评价方法将有助于今后研究APP生物学特性及基因功能。
[Abstract]:A ctinobacillus pleuropneumoniae (APP) is a pathogen causing porcine pleuropneumonia (Porcine pleuropneumonia). This disease is a serious * * * respiratory disease and is lethal. It has caused great economic losses to the pig industry. Up to now, 15 APP serotype.APP infections have been detected and caused. There are many virulence factors involved in the pathogenesis, including RTX toxin, capsular polysaccharide, lipopolysaccharide, adhesion factor, protease, outer membrane protein and transcription regulator. Through a variety of bioinformatics analysis software, we predicted that there were 60 kinds of lipoproteins in APP, and selected a special tandem repeat Q (E/D/P) QPK. Lip40. It was found that the repeats were different in different serotypes of APP strains. BioEdit (version 7.0) analysis showed that Lip40 had high similarity with many known lipoproteins and Tbp proteins. The results showed that Lip40 protein was composed of one p-tube (the beta-tube was composed of eight p-folds) and four p-folds. The three-dimensional structure overlap of Lip40 protein with APP TbpB N lobe and CLOBE was compared by WinCoot software. It was found that the three-dimensional structure of Lip40 protein was similar to that of APP TbpB N lobe and CLOB. The lip40 gene of APP SLW01 strain was cloned by PCR. After restriction endonuclease digestion, the target gene was inserted into pGEX-KG vector and transformed into E.coli BL21 competent cells. The recombinant protein rLip40 was obtained by IPTG induction. A hybridization band with a molecular weight of about 56 kDa appeared on the swimming track of rLip40, indicating that the positive serum against APP could be obtained. In addition, the polyclonal antibody against rLip40 was obtained by immunizing rabbits with rLip40 protein. The titer of the polyclonal antibody was 1:2 X 105, indicating that rLip40 protein could induce strong immune response in vivo, indicating that the Lip40 protein possessed the characteristics of immunoreactivity. In order to identify the subcellular localization of Lip40 protein, the subcellular localization of Lip40 protein was performed by immunoblotting with rabbit anti-rLip40 polyclonal antibody as the primary antibody. The results showed that the subcellular localization of Lip40 protein was in the adventitia, which was the same as the previous bioinformatics results. This study analyzed the expression of Lip40 protein under different stress conditions. The results showed that Lip40 protein was up-regulated at transcriptional and translation levels under high temperature, anaerobic and hypothermic stress conditions. Therefore, Lip40 protein was considered to be a multiple stress response factor, and Lip40 protein might be a virulent factor of APP, possibly a reference. Interaction with pathogen-environment and pathogen-host. This study analyzed the immunoprotective effect of Lip40 protein. Mice were immunized with rLip40 protein as an immunogen, and then infected with high pathogenic APP strain at lethal doses. The results showed that the protective rate of Lip40 protein in mice was 75%, although the protective rate was lower than that of ApxIA toxin protein. Lip40 protein can be used as a candidate protein for the development of high-efficiency APP vaccine. In this study, a reply mutant Clip40 was successfully constructed and its biological characteristics were preliminarily analyzed. JFF-lip40 could be inherited steadily in APP and had no significant effect on the growth ability of APP. In addition, strain APP SLW01 and delta-lip40 were highly sensitive to chloramphenicol, but the resistance of restored mutant CAlip40 to chloramphenicol was significantly stronger than that of the first two strains, suggesting that shuttle plasmid pJFF224-XN resistance gene could be stably expressed in APP strain. The successful construction of lip40 provides an effective material for analyzing the pathogenic mechanism of Lip40 protein. The construction system of complementary strains of APP and the evaluation method of APP resistance established in this study will be helpful to study the biological characteristics and gene function of APP in the future.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：2201388
[Abstract]:A ctinobacillus pleuropneumoniae (APP) is a pathogen causing porcine pleuropneumonia (Porcine pleuropneumonia). This disease is a serious * * * respiratory disease and is lethal. It has caused great economic losses to the pig industry. Up to now, 15 APP serotype.APP infections have been detected and caused. There are many virulence factors involved in the pathogenesis, including RTX toxin, capsular polysaccharide, lipopolysaccharide, adhesion factor, protease, outer membrane protein and transcription regulator. Through a variety of bioinformatics analysis software, we predicted that there were 60 kinds of lipoproteins in APP, and selected a special tandem repeat Q (E/D/P) QPK. Lip40. It was found that the repeats were different in different serotypes of APP strains. BioEdit (version 7.0) analysis showed that Lip40 had high similarity with many known lipoproteins and Tbp proteins. The results showed that Lip40 protein was composed of one p-tube (the beta-tube was composed of eight p-folds) and four p-folds. The three-dimensional structure overlap of Lip40 protein with APP TbpB N lobe and CLOBE was compared by WinCoot software. It was found that the three-dimensional structure of Lip40 protein was similar to that of APP TbpB N lobe and CLOB. The lip40 gene of APP SLW01 strain was cloned by PCR. After restriction endonuclease digestion, the target gene was inserted into pGEX-KG vector and transformed into E.coli BL21 competent cells. The recombinant protein rLip40 was obtained by IPTG induction. A hybridization band with a molecular weight of about 56 kDa appeared on the swimming track of rLip40, indicating that the positive serum against APP could be obtained. In addition, the polyclonal antibody against rLip40 was obtained by immunizing rabbits with rLip40 protein. The titer of the polyclonal antibody was 1:2 X 105, indicating that rLip40 protein could induce strong immune response in vivo, indicating that the Lip40 protein possessed the characteristics of immunoreactivity. In order to identify the subcellular localization of Lip40 protein, the subcellular localization of Lip40 protein was performed by immunoblotting with rabbit anti-rLip40 polyclonal antibody as the primary antibody. The results showed that the subcellular localization of Lip40 protein was in the adventitia, which was the same as the previous bioinformatics results. This study analyzed the expression of Lip40 protein under different stress conditions. The results showed that Lip40 protein was up-regulated at transcriptional and translation levels under high temperature, anaerobic and hypothermic stress conditions. Therefore, Lip40 protein was considered to be a multiple stress response factor, and Lip40 protein might be a virulent factor of APP, possibly a reference. Interaction with pathogen-environment and pathogen-host. This study analyzed the immunoprotective effect of Lip40 protein. Mice were immunized with rLip40 protein as an immunogen, and then infected with high pathogenic APP strain at lethal doses. The results showed that the protective rate of Lip40 protein in mice was 75%, although the protective rate was lower than that of ApxIA toxin protein. Lip40 protein can be used as a candidate protein for the development of high-efficiency APP vaccine. In this study, a reply mutant Clip40 was successfully constructed and its biological characteristics were preliminarily analyzed. JFF-lip40 could be inherited steadily in APP and had no significant effect on the growth ability of APP. In addition, strain APP SLW01 and delta-lip40 were highly sensitive to chloramphenicol, but the resistance of restored mutant CAlip40 to chloramphenicol was significantly stronger than that of the first two strains, suggesting that shuttle plasmid pJFF224-XN resistance gene could be stably expressed in APP strain. The successful construction of lip40 provides an effective material for analyzing the pathogenic mechanism of Lip40 protein. The construction system of complementary strains of APP and the evaluation method of APP resistance established in this study will be helpful to study the biological characteristics and gene function of APP in the future.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
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2 蔡宝祥;猪传染性胸膜肺炎的诊断与防治[J];辽宁畜牧兽医;1996年03期
，本文编号：2201388
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